EP3166629B1 - Emd mit niedriger viskosität - Google Patents

Emd mit niedriger viskosität Download PDF

Info

Publication number
EP3166629B1
EP3166629B1 EP15738878.6A EP15738878A EP3166629B1 EP 3166629 B1 EP3166629 B1 EP 3166629B1 EP 15738878 A EP15738878 A EP 15738878A EP 3166629 B1 EP3166629 B1 EP 3166629B1
Authority
EP
European Patent Office
Prior art keywords
bone
graft material
bone graft
composition
emd
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP15738878.6A
Other languages
English (en)
French (fr)
Other versions
EP3166629A1 (de
Inventor
Alessandra APICELLA
Peggy HEUNEMANN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Straumann Holding AG
Original Assignee
Straumann Holding AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Straumann Holding AG filed Critical Straumann Holding AG
Publication of EP3166629A1 publication Critical patent/EP3166629A1/de
Application granted granted Critical
Publication of EP3166629B1 publication Critical patent/EP3166629B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

Definitions

  • the present invention relates to the field of bone graft materials for implantation into patients in need thereof and their bioactivation.
  • the present invention further relates to a low viscosity composition comprising active enamel substances, which is herein used for bioactivating bone graft materials, and to a kit comprising said bone graft material in combination with said low viscosity composition comprising active enamel substances, as well as to the bioactivated bone graft material as such.
  • Periodontal tissue diseases are defined as an inflammatory condition of the gum and the bone support surrounding the teeth, often leading to complications, such as to bone degradation, which in turn can impend the correct placement and fixation of dental implants after tooth loss.
  • Bone grafting is a surgical procedure that replaces missing bone after bone degradations and/or in order to repair bone fractures that are extremely complex, pose a significant health risk to the patient, and/or fail to heal properly. Bone grafting is currently employed to remedy bone degradation coursed by periodontal tissue diseases with varying success rates.
  • Bone substitutes are in general increasingly used in surgery as over two millions bone grafting procedures are performed worldwide per year. Autografts are still widely preferred for bone substitution, though the morbidity and the inherent limited availability are the main limitations. Allografts, i.e. banked bone, are osteoconductive and weakly osteoinductive, though there are still concerns about the residual infective risks, costs and donor availability issues. As an alternative, xenograft substitutes are cheaper, but their use provides contrasting results, so far. Often, bovine bone is used to replace missing bone structure; the major advantage is that larger quantities of bone can be easily acquired compared to bone of human origin.
  • Ceramic-based synthetic bone substitutes are alternatively based on hydroxyapatite (HA) and tricalcium phosphates, and are widely used in the clinical practice. Indeed, despite being completely resorbable and weaker than cortical bone, they have exhaustively proven to be effective.
  • Biomimetic HAs are the evolution of traditional HA and contain ions (carbonates, Si, Sr, Fl, Mg) that mimic natural HA (biomimetic HA).
  • Injectable cements represent another evolution, enabling mininvasive techniques.
  • Bone morphogenetic proteins namely BMP2 and 7 are the only bone inducing growth factors approved for human use in spine surgery and for the treatment of tibial nonunion.
  • osteoconduction guiding the reparative growth of the natural bone
  • osteoinduction encouraging undifferentiated cells to become active osteoblasts
  • osteogenesis living bone cells in the graft material contribute to bone remodeling
  • Bone graft materials are autografts, allografts, xenografts or synthetic grafts.
  • Autologous (or autogenous) bone grafting involves utilizing bone obtained from the same individual receiving the graft.
  • Bone can be harvested from non-essential bones, such as the iliac crest or the fibula, the chin, the ribs, the mandible and even parts of the skull.
  • Autogenous bone possesses all the properties essential for bone formation: it is osteoconductive and osteoinductive, and it houses growth factors and osteogenic cells with no associated immune or infective-related risks.
  • Autologous bone fractions are slowly replaced by newly formed host bone.
  • the disadvantages of autografts are that a surgical donor site is required, leading to possible post-operative pain and complications. Also, they carry a likelihood of blood loss or hematomas, infection, fracture, neurovascular injury, as well as cosmetic deformity, at the explantation site, and longer operative time.
  • Allograft biobanked bone represents a suitable alternative to autogenous bone, being derived from humans as well.
  • Allograft bone can be collected from either living donors (patients total hip replacement surgery) or nonliving donors and must be processed within a bone tissue bank.
  • Donor bone is osteoconductive, weakly osteoinductive (growth factors may still be present, depending on the processing).
  • allografts often require sterilization (gamma-irradiation), with detrimental effects on mechanical properties of bone, and deactivation of proteins normally found in healthy bone.
  • transplants The limits of such transplants are costs, laborious procedure (tissue processing, harvesting), mechanical resistance (in freeze dried and irradiated), limited osteoinduction and risk of infection.
  • Xenograft bone substitutes have their origin from a species other than human, such as bovine bone (or porcine bone) which can be freeze dried or demineralized and deproteinized. Xenografts are usually only distributed as a calcified matrix. Coral based xenografts are mainly calcium carbonate (and an important proportion of fluorides, useful in the context of grafting to promote bone development) while natural human bone is made of hydroxyapatite along with calcium phosphate and carbonate.
  • the coral material is thus either transformed industrially into hydroxyapatite through a hydrothermal process, yielding to a non-resorbable xenograft, or simply the process is omitted and the coralline material remains in its calcium carbonate state for better resorption of the graft by the natural bone.
  • the coral xenograft is then saturated with growth enhancing gels and solutions. Xenografts have given good results in dentistry, but scarce validation is available in orthopedics.
  • BioOss® is a natural product with bovine origin. It is deproteinized and sintered. The material's total porosity is between 70 - 75% with a particle size of 250 - 1000 ⁇ m.
  • the advantages are the easy availability, the osteoconductivity, the good mechanical properties and low costs.
  • synthetic bone substitutes are calcium based substitutes, in particular, a mix of HA (Hydroxyapatite) and TCP (Tricalcium phosphate), HA is a relatively inert substance that is retained " in vivo " for prolonged periods of time, whereas the more porous TCP typically undergoes biodegradation within 6 weeks of its introduction into the area of bone formation. HA achieves very high mechanical strength, while TCP has lower mechanical qualities.
  • the base is a biphasic calcium phosphate, which combines 40-60 % TCP with 60-40 % HA, yielding a more physiological balance between mechanical support and bone resorption.
  • Synthetic bone grafts are widely known and are proven to be safe and effective in bone substitution.
  • HA-TCP materials are available in form of blocks, granules and injectable kits.
  • the pore size varies between different materials but is generally within the range of 0.1 to 1000 ⁇ m, such as 100-800 ⁇ m. Pore interconnectivity is necessary for bone ingrowth.
  • the strength is variable between 10 and 60 MP, which is lower than cortical bone compression strength (150-200 MP), which is one of the major limit of ceramic based biomaterials.
  • An exemplary HA-TCP material includes a porous biphasic synthetic bone-graft substitute in granulated form, herein denoted Oss. It consists of biphasic calcium phosphate, a composite of 10% hydroxyapatite and 90% ⁇ -tricaicium phosphate. The pore size is 0.1 - 1000 ⁇ m. The total porosity in this material is about 50-85%, such as 65 ⁇ 15% or 70 ⁇ 15%.
  • HA-TCP material is Straumann BoneCeramic® (Straumann AG, Basel, Switzerland) which is a synthetic bone-graft substitute designed for augmenting bone. It consists of biphasic calcium phosphate with a composite of 60% hydroxyapatite and 40% ⁇ -tricalcium phosphate. BoneCeramic® is 90% porous with interconnected pores of 100-500 ⁇ m in diameter.
  • Botiss maxresorb® (Botiss dental GmbH, Berlin, Germany) which is a synthetic bone-graft substitute designed for augmenting bone. It consists of biphasic calcium phosphate with a composite of 60% hydroxyapatite and 40% ⁇ -tricalcium phosphate. Maxresorb® is 80% porous with interconnected pores of 200-800 ⁇ m in diameter and micropores having a diameter of 1-10 ⁇ m.
  • HA Hydroxyapatite
  • TCP Hydroxyapatite
  • HA is the primary mineral component of teeth and bone.
  • HA ceramics come in both naturally and synthetic forms.
  • HA and TCP ceramics are manufactured in a variety of forms including granules and porous blocks. TCP is more soluble than HA.
  • HA accounts for nearly 70 % of the mineral content of teeth and bone, the occurring HA in the human body exists in a substituted form.
  • Carbonate, silicates, and magnesium among other ions may replace hydroxyl or phosphate groups of the apatite structure.
  • Investigators have attempted to produce HA that more closely resembles the mineral content of native bone, enhancing bioactivity and osteoconduction (Biomimetic ceramic substitutes).
  • CPC Calcium phosphate cements
  • the cements are a white powder, consisting of calcium phosphate, that when mixed with a liquid, forms a workable paste which can be shaped during surgery to fit the contours of bone loss.
  • the cements harden within 20 min.
  • the hardening reaction, which forms nanocrystalline hydroxyapatite (HA) is isothermic and occurs at physiologic pH so tissue damage does not occur during the setting reaction.
  • CPCs were FDA approved for the treatment of non-load-bearing bone defects in 1996.
  • HA is the primary inorganic component of natural bone which makes the hardened cement biocompatible and osteoconductive. Over time, CPCs are gradually resorbed and replaced with new bone.
  • CPCs are brittle, they are used for non-load-bearing applications such as dental, cranio-facial and orthopedic applications.
  • CPCs have two significant advantages over pre-formed, sintered ceramics. First, the CPCs paste can be sculpted during surgery to fit the cavities. Second, the nanocrystalline hydroxyapatite structure of the CPC makes it osteoconductive causing it to be gradually resorbed and replaced with new bone.
  • CS Calcium sulphate
  • Polymers have physical, mechanical, and chemical properties completely different from the other bone substitutes.
  • the polymers can be divided into natural polymers and synthetic polymers. These, in turn, can be divided further into degradable and nondegradable types.
  • Degradable synthetic polymers are resorbed by the body. The benefit is that they enhance healing without remaining foreign bodies.
  • Degradable polymers such as polylactic acid and poly(lactic-co-glycolic acid) have been used as standalone devices and as extenders of autografts and allografts.
  • Bone is mainly made of collagen (Col) and carbonate substituted hydroxyapatite (HA). Actually it is possible to obtain Col-HA by a self-assembling process on a nanometric scale.
  • an implant manufactured from such components is likely to behave better than other bone substitutes made as monolithic devices. Indeed, both collagen type I and hydroxyapatite were found to enhance osteoblast differentiation, but combined together, they were shown to accelerate osteogenesis.
  • bone-inducing growth factors are currently known in the field of the art, such as bone morphogenetic proteins (BMP), insulin growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (FGF), able to stimulate activation and migration of osteogenic stem cells and progenitor cells, and to induce revascularization.
  • BMP bone morphogenetic proteins
  • IGF insulin growth factor
  • TGF transforming growth factor
  • FGF fibroblast growth factor
  • the challenge to tissue engineers is to design and develop temporary bone scaffolds which deliver bioactive molecules and drugs or cells to the injury site and hence extend its biological functionality (accelerate healing and tissue regeneration while simultaneously preventing pathology).
  • mimicking the geometric architecture of bone in a synthetic scaffold has been shown to promote favorable cellular activity, the overall capacity for a scaffold to direct cell behavior can be substantially improved through the controlled delivery of bio specific cues.
  • Administration of growth factors and other bioactive molecules to promote bone formation and repair has achieved promising results in several preclinical and clinical models.
  • the efficacy of the delivery vehicle relies on its ability to provide the appropriate dose over the appropriate therapeutic time.
  • the presentation of bioactive molecules or drugs must be finely tuned to dynamically match the physiological needs of the tissue as it regenerates.
  • New strategies work to encapsulate and release drugs which prevent pathologies that can occur post implantation of a synthetic scaffold.
  • drugs have been encapsulated and released from biodegradable polymer scaffolds including antibiotics, DNA, RNA, cathepsin inhibitors, chitin, chemotherapeutics, bisphosphonates, statins, sodium fluoride, dihydropyridine, and many others.
  • researchers are aggressively pursuing strategies to deliver antibiotics locally to the site of injury/surgery. Although local delivery of antibiotics has a very promising outlook, there remains a number of challenges (such as antibiotic stability within the scaffold and antibiotic deactivation during fabrication), which still need to be addressed.
  • Emdogain® a gel that is injectable by means of a syringe to the site of a bone defect
  • This gel consists of two components, propylene glycol alginate (PGA) and Enamel Matrix Derivative (EMD).
  • PGA propylene glycol alginate
  • EMD Enamel Matrix Derivative
  • PGA has a structural role and acts as a carrier
  • EMD is the active component that favours the regeneration of the diseased periodontal tissue by mediating the formation of acellular cementum at the root of the tooth and providing a foundation for the growth of the tissue associated with functional attachment.
  • the pH tends to strive to the physiological value and when it reaches a value of 6, it causes EMD to precipitate.
  • the osteoblast and cementoblast cells are enticed by the natural cocktail of isolated enamel matrix proteins to proliferate and cause the ligament extension from the gingival wall into the intrabony gum.
  • Straumann® Emdogain® is a commercially available product composed of Propylene Glycol Alginate (PGA) and porcine Enamel Matrix Derivative (EMD) proteins.
  • the PGA employed in the manufacture of Straumann® Emdogain® has a viscosity of 50-175 mPa ⁇ s (EMD in a 2 % PGA aqueous solution 22°C (Brookfield viscosity)).
  • the composition Straumann® EMDOGAIN® itself, displays a viscosity of 3.0 Pa ⁇ s (3000 mPa ⁇ s at 22°C). Upon dissolving alginates in water, the molecules hydrate and the solution gains viscosity.
  • the viscosity of an alginate solution depends on the concentration of alginate and the length of the alginate molecules, i.e. the number of monomer units in the chains. In general, the longer the chains, the higher the viscosity at similar concentrations.
  • the dissolved molecules are not completely flexible; rotation around the glycosidic linkages in the G-block regions is somewhat hindered, resulting in a stiffening of the chain. Solutions of stiff macromolecules are highly viscous.
  • EMD is nearly 90% composed of amelogenin
  • These are generally only marginally stable and sensitive to environmental stresses such as temperature or pH changes, which may lead to an irreversible unfolding or misfolding of the proteins. This in turn leads to the proteins forming large unfolded aggregates that are unable to carry out their function as tissue healing inducer.
  • EMD periodontal regeneration following EMD application
  • its low viscosity is of concern.
  • use of EMD with a porous interconnected scaffold may be preferable.
  • the scaffold should allow cell migration and proliferation, but also subsequently be assimilated by the surrounding.
  • amelogenin the main active ingredient of EMD folds into a complex tertiary conformational structure and several units need to assemble into a macrostructure for the protein to assert its biological activity.
  • a biocompatible material developed from a natural bone mineral of bovine origin, in one embodiment available as granules of spongious bone having a bimodal pore distribution, as well as a porous synthetic bone substitute, were for the first time successfully bioactivated with a low viscosity composition comprising EMD as is described in the following.
  • the present document is directed to a composition for bioactivating a bone graft material, said composition comprises isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 mPa ⁇ s at 22°C (room temperature).
  • the pharmaceutical carrier is preferably not propylene glycol alginate (PGA).
  • the concentration of enamel matrix proteins in the composition may be about 29-31 mg/ml, such as about 29, 30 or 31 mg/ml.
  • the suitable pharmaceutical carrier may e.g. be acetic acid or PBS. In a preferred embodiment, the pharmaceutical carrier is acetic acid.
  • the present document is also directed to the use of a composition as defined herein for bioactivating a bone graft material for improved bone regeneration.
  • the present document is thus also directed to a process for preparing a bioactivated bone graft material for improved bone regeneration, comprising
  • the pH of the composition may be between pH 3.8 - 4.2 before mixing components a) and b).
  • the total porosity of the bone graft material may be about 70-98%, such as 70%, 75%, 80%, 85%, 90.0%, 91.2%, 92%, 93% or 95%.
  • the composition comprising isolated enamel matrix proteins used in step b) may comprise isolated enamel matrix proteins at a concentration of about 29-31 mg/ml, such as at a concentration of about 29, 30 or 31 mg/ml.
  • the suitable pharmaceutical carrier of the composition comprising isolated enamel matrix proteins used in step b) may e.g. be acetic acid or PBS.
  • the bone graft material may e.g. be selected from the group consisting of natural bone, synthetic bone and scaffolds, such as natural bone and synthetic bone material.
  • the present document is also directed to a bioactivated bone graft material for improved bone regeneration which is prepared using a process comprising the steps of:
  • the present document is also directed to bioactivated porous bone graft material for improved bone regeneration, wherein at least 50-90% of the isolated enamel matrix proteins comprised in and/or on the bioactivated bone graft material are assembled amelogenin nanospheres, which precipitate in a main peak centered around 1500 nm, as measured by DLS.
  • the bone graft material may be selected from the group comprising a bovine xenograft and a synthetic bone graft material.
  • the bone graft material may also be in the form of granules.
  • the overall porosity of the bone graft material is at least 70%.
  • the present document is also directed to a kit for preparing a bioactivated bone graft material for improved bone regeneration as defined herein, said kit comprising at least two components; 1) a porous bone graft material, and 2) a composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH3.8 - 4.2, and a viscosity of less than 50 mPa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT).
  • RT viscosity
  • the present document is also directed to a bioactivated bone graft material as defined herein for use in medicine.
  • the present document is also directed to a bioactivated bone graft material for improved bone regeneration as defined herein for use in improving bone regeneration.
  • the present document is also directed to the use of a composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT) for manufacturing a bioactivated bone graft material as defined herein for improving bone regeneration.
  • RT viscosity
  • the present document is also directed to a method for improving bone regeneration in a patient in need thereof comprising implanting a bioactivated bone graft material as defined herein.
  • the present invention for the first time discloses a low viscosity composition comprising enamel matrix derivative (EMD) which, when applied to a porous bone graft material, improves and/or enhances bioactivity and osteoconduction of said bone graft material.
  • EMD enamel matrix derivative
  • the present invention consequently also for the first time discloses a bioactivated bone graft material with enhanced bioactivity and/or improved osteoconduction promoting properties comprising enamel matrix derivative (EMD).
  • EMD enamel matrix derivative
  • a kit comprising a) a low viscosity composition comprising enamel matrix derivative (EMD) together with b) a porous bone graft material, to be combined immediately before the placing of the bioactivated bone graft material by the skilled practitioner.
  • a bioactivated bone graft material, soaked in and/or coated with a low viscosity composition comprising enamel matrix derivative (EMD) is provided ready for use.
  • bone substitutes obtained from the mineral part of bovine bone are demonstrated to fulfill both physical and biological requirements for a new, easy to apply injectable scaffold for use in combination with a low viscosity composition comprising enamel matrix derivative (EMD).
  • EMD enamel matrix derivative
  • biocompatible material developed from a natural bone mineral of bovine origin (Bio-Oss®), as well as one of synthetic origin (Oss) consisting of biphasic calcium phosphate, a composite of 10% hydroxyapatite and 90% ⁇ -tricaicium phosphate, having a bimodal pore distribution, was successfully bioactivated with a low viscosity composition comprising EMD.
  • the bioactivated scaffold material in this particular embodiment resembles the human bone, and its interconnecting pores permit the formation and ingrowth of new tissue.
  • the first experiments done consisted in observing the behavior and characterizing of different combinations of solutions containing EMD diluted in acetic acid and Bio-Oss®, a biocompatible material, developed from a natural bone mineral of bovine origin. Afterwards, the same experiments were performed replacing Bio-Oss® with Oss (a synthetic bone substitute material), which is another scaffold material mainly composed of hydroxyapatite / TCP granules. Parameters as the influence of time and the initial pH of the EMD solutions were investigated by fluorescence, Dynamic Light Scattering (DLS), and Scanning Electron Microscopy (SEM). These analyses permitted obtaining useful information on how the aggregation and precipitation processes evolve in time, on the size and morphology of the formed aggregates, and about their coverage on the Bio-Oss® or Oss granules.
  • DLS Dynamic Light Scattering
  • SEM Scanning Electron Microscopy
  • EMD was dissolved in buffer systems at different pH, mixed with bovine bone grafts (1:1), and the pH and viscosity changes recorded.
  • the Brunner-Emmer-Teller (BET) technique was used to measure bone graft porosity and specific surface area.
  • EMD aggregation before and after bone graft addition was monitored by fluorescence measurements and aggregate/precipitate sizes were determined by dynamic light scattering (DLS).
  • DLS dynamic light scattering
  • pH 4 Below pH 4, precipitation was delayed, and partial unfolding of the EMD resulted in a shift in the size distribution of the aggregates to higher values. pH 4 therefore provided the best compromise in properties, the delay in EMD precipitation allowing sufficient time for application, while being short enough for the folded EMD conformation to be maintained and to ensure homogenous coverage of bone graft surface. Different aggregation rates and precipitate sizes were observed depending on the initial pH of the EMD solutions and the overall porosity of the bone graft material used. There is no reason to speculate that the actual origin of the material is of any crucial importance, but instead the favourable precipitation, aggregation and/or agglomeration seems at least in part to occur in relation to and/or be improved by the porosity of the material used.
  • the present invention for the first time discloses a low viscosity composition comprising enamel matrix derivative (EMD) which, when applied to a porous bone graft material, improves and/or enhances bioactivity and osteoconduction of said bone graft material.
  • EMD enamel matrix derivative
  • the present invention for the first time discloses a composition for bioactivating a bone graft material, comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 mPa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT).
  • Said composition is in the present context used for bioactivating a porous bone graft material for improved bone regeneration.
  • Enamel matrix proteins, present in the enamel matrix, are most well-known as precursors to enamel. Prior to cementum formation, enamel matrix proteins are deposited on the root surface at the apical end of the developing tooth-root. There is evidence that the deposited enamel matrix is the initiating factor for the formation of cementum. Again, the formation of cementum in itself is associated with the development of the periodontal ligament and the alveolar bone. Enamel matrix proteins can therefore promote periodontal regeneration through mimicking the natural attachment development in the tooth ( Gestrelius S, Lyngstadaas SP, Hammarström L. Emdogain - periodontal regeneration based on biomimicry. Clin Oral Invest 4:120-125 (2000 )).
  • Isolated enamel matrix proteins are able to induce not only one, but an orchestrated cascade of factors, naturally found in tissues developing adjacent to the enamel matrix. They mimic the natural environment of a developing tissue and thus mimic a natural stimulation for tissue regeneration, cell differentiation and/or maturation.
  • Enamel matrix derivative in the form of a purified acid extract of proteins from pig enamel matrix, has previously been successfully employed to restore functional periodontal ligament, cementum and alveolar bone in patients with severe tooth attachment loss ( Hammarström et al., 1997, Journal of Clinical Periodontology 24, 658-668 ).
  • Enamel Matrix Derivative (EMD) proteins and enamel matrix proteins are widely used in clinical dentistry because of their ability to promote regeneration of soft and hard tissues and to reduce inflammation and infections.
  • Purified Enamel Matrix Derivative (EMD) proteins contain 3 major protein fractions which are separable by High Pressure Liquid Chromatography (HPLC). These fractions are named fraction A, B and C, respectively.
  • a typical weight ratio of the isolated and/or purified proteins is about 80/8/12 between the main protein peaks at 20, 14 and 5 kDa, respectively.
  • the fraction C typically has a molecular weight of between approximately 3, 5 and 5 kDa, such as approximately 5 kDa, 4 kDa and 3,5 kDa, as determined by SDS PAGE electrophoresis.
  • the fraction A typically has a molecular weight of approximately 20 kDa, as determined by SDS PAGE electrophoresis.
  • the fraction B typically has a molecular weight of between approximately 6 kDa and 15 kDa, such as approximately 15 kDa, 12 kDa, 10 kDa and 6 kDa, as determined by SDS PAGE electrophoresis.
  • EMD proteins and/or enamel matrix proteins are composed of a number of proteins, such as amelogenin, enamelin, tuft protein, proteases, and albumin.
  • Amelogenin a major constituent of EMD proteins and/or enamel matrix proteins (up to approximately 90%), are a family of hydrophobic proteins derivable from a single gene by alternative splicing and controlled post secretory processing. They are highly conserved throughout vertebrate evolution and demonstrate a high overall level of sequence homology among all higher vertebrates examined (80%). In fact, the sequences of porcine and human amelogenin gene transcript differ only in 4% of the bases.
  • enamel matrix proteins or EMD proteins although of porcine origin, are considered "seif" when encountered in the human body and can promote dental regeneration in humans without triggering allergic responses or other undesirable reactions.
  • purified Enamel Matrix Derivative (EMD) proteins are thus defined as enamel matrix proteins comprising at least 60-70% amelogenin, such as at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 %, with a molecular weight of about 20-25 kDa, such as 20, 21, 22, 23, 24, or 25 kDa, or such as between 20-22, 20-24, or 20-23 kDa.
  • the weight ratio of the purified and/or isolated enamel matrix proteins is about 80/8/12, such as 75-85/5-12/5-15, or such as at least 80%, at least 8%, and at least 5%, between the main protein peaks of fraction A, B and C, respectively.
  • Approximately 60-90%, such as at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 80, 70-90, 60-70, 70-80, or 80-90% of the purified and/or isolated enamel matrix proteins are amelogenin and/or fragments or derivatives of amelogenin.
  • Methods to determine identity and similarity are codified in publicly available programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al (1994)) BLASTP, BLASTN, and FASTA (Altschul, S.F. et al (1990)).
  • the BLASTX program is publicly available from NCBI and other sources ( BLAST Manual, Altschul, S.F. et al, Altschul, S.F. et al (1990)).
  • Another preferred example is Clustal W (http://www.ebi.ac.uk/clustalw/) .
  • Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used.
  • the amino acids in an EMD protein and/or enamel matrix protein may further be modified in terms of chemistry, isometry or in any other way as long as the sequences of the protein is intact. Modifications of the amino acids of the EMD protein and/or enamel matrix protein may increase the activity, stability, biocompatibility or clinical performance of the proteins, or reduce toxicity and adverse reactions to the proteins. Examples of chemical modifications include, but are not limited to, glycosylation and methylation.
  • the amino acids may also be of all different types of stereoisomeric forms, such as D or L forms of amino acids, or S or R isomers.
  • the amino acids in an EMD protein and/or enamel matrix protein of the invention may also be replaced by synthetic analogues thereof. The use of synthetic analogues may e.g.
  • unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-l-phenylalanine*, L-allyl-glycine*, ⁇ -alanine*, L-a-amino butyric acid*, L-g-amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxy
  • EMD proteins and/or enamel matrix proteins may further comprise N- and/or C-terminal tags comprising the amino acids His and/or Met.
  • the EMD proteins and/or enamel matrix proteins are produced instead of isolated from a natural source, e.g. by synthetic production or biosynthesis.
  • the EMD proteins and/or enamel matrix proteins, or fragments thereof may be produced by any known method for production of peptides, such as synthetic production by chemical synthesis. Synthetic production also allows the use of amino acid analogues which may improve the stability of the proteins or fragments produced. The skilled person knows the methods that are available for the synthesis of an amino acid sequence.
  • bioproduction may be used as a method for producing the EMD proteins and/or enamel matrix proteins, or fragments thereof.
  • Bioproduction means the production of an amino acid sequence in a biological system, such as a cell culture or in microbial cells, e.g. bacterial cells.
  • the present invention relates to a composition
  • a composition comprising enamel matrix proteins and/or enamel matrix derivative (EMD) proteins, wherein at least 60-70%, such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70%, of the proteins have a molecular weight between 16-40 kDa, such as above 20 kDa, such as 16, 17, 18 or 19 kDa.
  • EMD enamel matrix derivative
  • composition comprising isolated enamel matrix proteins used in step b) of the process described in the present invention comprises isolated enamel matrix proteins at a concentration of 0.01 to 100 mg/ml, such as of 0.3 mg/ml-100 mg/ml, or of 29-31 mg/ml, such as at a concentration of 29, 30 or 31 mg/ml.
  • concentration of the EMD proteins and/or enamel matrix proteins in a composition according to the invention is around 29 mg/ml.
  • EMD proteins and/or enamel matrix proteins are applied to the porous bone graft material in a composition according to the present invention, comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT), the EMD proteins and/or enamel matrix proteins precipitate in a folded state, preserving their regenerative properties, and coat the bone graft homogeneously to give a viscous, biocompatible scaffold, with the potential to fill periodontal defects and prevent flap collapse.
  • RT m Pa ⁇ s at 22°C
  • composition may be formulated according to conventional pharmaceutical practice, see, e.g., " Remington's Pharmaceutical Sciences” and “ Encyclopedia of Pharmaceutical Technology”, edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988 .
  • pharmaceutically or cosmetically acceptable excipient, carrier and/or diluent is a substance which is substantially harmless to the individual to which the formulation is to be administered.
  • excipient, carrier and/or diluent normally fulfills the requirements given by the national health authorities.
  • Official pharmacopoeias such as e.g. the British Pharmacopoeia, the United States of America Pharmacopoeia and The European Pharmacopoeia set standards for pharmaceutically acceptable excipients.
  • the suitable pharmaceutical carrier of the present invention may include solvents, buffering agents, preservatives, humectants, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gel-forming agents, ointment bases, penetration enhancers, perfumes, and skin protective agents.
  • buffering agents are e.g. citric acid, acetic acid, tartaric acid, lactic acid, hydrogenphosphoric acid, etc.
  • the suitable pharmaceutical carrier of the composition comprising isolated enamel matrix proteins is to be selected from the group consisting of acetic acid and PBS. In a presently preferred embodiment, the suitable pharmaceutical carrier of the composition comprising isolated enamel matrix proteins is acetic acid.
  • the carrier does not comprise or consist of propylene glycol alginate (PGA).
  • PGA propylene glycol alginate
  • the composition of the present invention comprising isolated enamel matrix proteins may further include arginine.
  • arginine In particular in a concentration range of 700 mM or lower, such as 10-700 mM, 100-700 mM or 200-700 mM is preferred, in particular when the concentration of EMD is at least 20 mg/ml, such as about 29-31 mg/ml, such as about 31 mg/ml.
  • Different aggregation rates and precipitation sizes depend at least partially on the initial pH of the composition.
  • pH 4 a range of pH between pH 3.8-4.2 and in particular pH 4 is identified as a critical threshold above which EMD aggregation and precipitation occurs immediately after bone graft addition, and agglomerates no longer completely cover the bone graft surface.
  • pH 4 a critical threshold above which EMD aggregation and precipitation occurs immediately after bone graft addition, and agglomerates no longer completely cover the bone graft surface.
  • pH 4 a range of pH between pH 3.8-4.2 and in particular pH 4 is identified as a critical threshold above which EMD aggregation and precipitation occurs immediately after bone graft addition, and agglomerates no longer completely cover the bone graft surface.
  • pH 4 therefore provides the best compromise in properties, the delay in EMD precipitation allowing sufficient time for application, while being short enough for the folded EMD conformation to be maintained and to ensure homogenous coverage of bone graft surface.
  • the present invention provides for a composition for bioactivating a bone graft material, comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT) and a pH between pH 3.8 - 4.2, such as selected from the group consisting of a pH between 3.8-4-1, 3.8-4.0, 3.8-3.9, 3.9-4.2, 3.9-4.1, 3.9-4.0, 4.0-4.1, 4.0-4.2, and 4.1-4.2.
  • said composition for bioactivating a bone graft material comprising isolated enamel matrix proteins has a pH of 3.8, 3.9, 4.0, 4.1 or 4.2.
  • the present invention also provides for a use of a composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, for bioactivating a bone graft material for improved bone regeneration, said composition having a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT) and a pH between pH 3.8 - 4.2.
  • the pH of the composition is between pH3.8 - 4.2 before mixing components a) and b).
  • Different aggregation rates and precipitation sizes depend at least partially on the initial viscosity of the composition.
  • a bone graft is a porous material.
  • the surface of a given bone graft is in general distributed throughout the bone graft, as well as at the periphery of the graft.
  • a suitably low viscosity therefore allows for EMD (amelogenin) aggregation, precipitation and/or agglomeration to occur on the complete bone graft surface and not only on the peripheral surfaces of the graft. This ensures homogenous coverage of a given bone graft surface.
  • the present invention relates to a low viscosity composition
  • a low viscosity composition comprising isolated enamel matrix proteins.
  • a low viscosity composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2.
  • the low viscosity composition according to the present invention has a viscosity that is substantially lower than the viscosity of the commercially available of EMD formulated in PGA, which is well known to be 3000 Pa ⁇ s at 22°C, comprising EMD in a 2% aqueous solution of PGA with a viscosity between 50-175 mPa ⁇ s at 22°C (RT).
  • the low viscosity composition according to the present invention typically has a viscosity of less than 50 mPa ⁇ s (EMD in an aqueous solution at 22°C), such as less than 50, 40, 30, 20, 10, 5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 mPa ⁇ s at 22°C (RT).
  • EMD mPa ⁇ s
  • RT mPa ⁇ s
  • the low viscosity composition according to the present invention typically has a viscosity selected from the group consisting of 50-1.0 mPa ⁇ s at 22°C (RT), 50-10 mPa ⁇ s at 22°C (RT), 50-1.7 mPa ⁇ s at 22°C (RT), 30-10 mPa ⁇ s at 22°C (RT), 30 - 1.7 mPa ⁇ s at 22°C (RT), 30 - 1.0 mPa ⁇ s at 22°C (RT), 10 - 2 mPa ⁇ s at 22°C (RT), 10- 1.7 mPa ⁇ s at 22°C (RT), 10 - 1.0 mPa ⁇ s at 22°C (RT), 5 - 1.7 mPa ⁇ s at 22°C (RT), 2.5-1.2 mPa ⁇ s at 22°C, 2 - 1.0 mPa ⁇ s at 22°C (RT) and 1.8 - 1.0 mPa ⁇ s at 22°C (RT), such as at the most 50, 45
  • the low viscosity composition according to the present invention has a viscosity that is particularly suitable for bioactivating a bone substitute with an overall porosity of at least 70%, i.e. a viscosity which is no more than 1.8 mPa ⁇ s at 22°C, such as no more than 1.7 mPa ⁇ s at 22°C, such as no more than 1.6 mPa ⁇ s at 22°C, such as no more than 1.5 mPa ⁇ s at 22°C, such as no more than 1.4 mPa ⁇ s at 22°C, such as no more than 1.3 mPa ⁇ s at 22°C, such as no more than 1.2 mPa ⁇ s at 22°C, such as no more than 1.1 mPa ⁇ s at 22°C, or such as no more than 1.0 mPa ⁇ s at 22°C, said composition comprising isolated enamel matrix proteins at a concentration of approximately 28-31 mg/ml in acetic acid and said composition having a pH between pH
  • a typical low viscosity composition of the present invention suitable for bioactivating a bone substitute with an overall porosity of at least 70%, has a viscosity of no more than 1.7 mPa ⁇ s at 22°C, comprises isolated enamel matrix proteins at a concentration of approximately 30 mg/ml in acetic acid and has a pH 4.0.
  • a low viscosity composition of the present invention suitable for bioactivating a bone substitute with an overall porosity of at least 70%, which comprises isolated enamel matrix proteins at a concentration of approximately 30 mg/ml in acetic acid and has a pH 4.0 and a viscosity between 2.5-1.2 mPa ⁇ s at 22°C.
  • the low viscosity composition according to the present invention is a composition for bioactivating a bone graft material, said composition comprises isolated enamel matrix proteins at a concentration of approximately 28-31 mg/ml in acetic acid, said composition having a pH between pH 3.8 - 4.2, and a viscosity of at the most 1.7 times the viscosity of water (measured as relative viscosity of liquids). With a given water viscosity of 0.9548 cp, the viscosity of said composition is 1.7 cp or less, such as 1.6 cp, 1.5 cp, 1.4 cp, 1.3 cp, 1.2 cp, 1.1 cp or less.
  • the substantially lower viscosity of the present composition comprising isolated enamel matrix proteins enables the composition to soak, coat and/or penetrate the porous bone graft material. Furthermore, the low viscosity of the composition comprising EMD favors amelogenin to precipitate and/or to agglomerate in a correct and/or coordinated fashion once it is applied onto or into the bone graft material.
  • bovine bone grafts and/or synthetic bone graft material such as synthetic bone grafts
  • a composition comprising isolated enamel matrix proteins in solution causes a pH increase and viscosity increase, which is shown to favor correct EMD aggregation and precipitation.
  • a low viscosity composition according to the present invention can further be characterized by a shear rate (s -1 ) which is 0.001 - 500.
  • the present invention relates to a bioactivated bone graft material for improved bone regeneration which is prepared using a low viscosity composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT).
  • RT m Pa ⁇ s at 22°C
  • Aggregation herein used exchangeable with protein aggregation, is a biological phenomenon in which proteins aggregate (i.e., accumulate and clump together) either intra- or extracellularly.
  • proteins After synthesis, proteins typically fold into a particular three-dimensional conformation: their native state. Only in their native state are they functional. This folding process is driven by the hydrophobic effect: a tendency for hydrophobic portions of the protein to shield itself from the hydrophilic interior of the cell by burying into the interior of the protein.
  • the exterior of a protein is typically hydrophilic, whereas the interior is typically hydrophobic.
  • Protein aggregation can occur at all steps in the manufacturing process (cell culture, purification and formulation), storage, distribution and handling of products. It results from various kinds of stress such as agitation and exposure to extremes of pH, temperature, ionic strength, or various interfaces (e.g. air-liquid interface). High protein concentrations (as in the case of some monoclonal antibody formulations) can further increase the likelihood of aggregation.
  • Protein aggregates span a broad size range, from small oligomers (nanometers) to insoluble micron-sized aggregates that can contain millions of monomer units.
  • Aggregation needs to be carefully characterized and controlled during development, manufacture and subsequent storage of a formulated product. Similarly, by monitoring the state of aggregation, modification or optimization of the production process can be achieved.
  • Agglomeration is in the present context used to describe the process of smaller particles coming together and forming a larger whole.
  • precipitation is herein used interchangeably with protein precipitation and describes the creation of a solid in a solution or inside another solid during a chemical reaction or by diffusion in a solid.
  • controlled aggregation and/or precipitation of enamel matrix proteins into correctly folded proteins having a biologically active conformation, as well as correct agglomeration of the isolated enamel matrix proteins into a biologically active macrostructure is achieved in a controlled manner on at least parts of the surface of a porous bone graft material, when a porous bone graft material according to the present invention is brought into contact with a low viscosity composition according to the present invention comprising the isolated enamel matrix proteins.
  • the present invention consequently discloses a bioactivated bone graft material with enhanced bioactivity and/or improved osteoconduction promoting properties.
  • said bone graft material displays enhanced biocompatibility after implantation.
  • the present invention provides for a bioactivated porous autologous graft, allograft, xenograft, and/or synthetic graft, comprising isolated enamel matrix proteins.
  • the term "bone graft" is used to describe autologous grafts, allografts, xenografts, or synthetic grafts with similar mechanical properties to bone.
  • Factors of importance for the biological behavior of a bone graft material include both physical and chemical factors. Physical factors of importance include form (particulate or bulk), porosity (density, pore size), surface area, surface charge and/or surface hydrophilicity, and crystallinity (crystal size, crystal perfection, and grain size).
  • Chemical factors include composition and ionic substitution in the material, while the biological factors include local declines of the pH, degree of bone contact, type of bone, species, age, gender, hormone levels, genetic predisposition etc.
  • the bone graft material should mimic the properties of natural bone in order to allow for a successful integration after implantation, e.g. in terms of how amelogenin binds to bone in vivo.
  • bone substitutes obtained from the mineral part of bovine bone are demonstrated to fulfill both physical and biological requirements for a new, easy to apply injectable scaffold for use in combination with a low viscosity composition comprising enamel matrix derivative (EMD).
  • EMD enamel matrix derivative
  • one factor that affects the agglomeration of amelogenin onto the surface of the material is the over-all porosity of the material, i.e. the total porosity including all kinds of pores, such as macro, micro and nano pores. Different aggregation rates and precipitate sizes depend on the initial pH of the EMD solutions, it's viscosity and the total porosity of the bone graft material used.
  • isolated enamel matrix protein aggregation and precipitation occurs in a window of time after bone graft addition, allowing for agglomerates of enamel matrix proteins, such as amelogenins, to homogenously cover at least parts of the bone graft surface, preferably the complete surface of the bone graft.
  • the practitioner must be allowed a certain delay in EMD precipitation allowing for sufficient time for application, while being short enough for the folded EMD conformation to be maintained and to ensure homogenous coverage of bone graft surface.
  • sufficient aggregation and/or precipitation and agglomeration of enamel matrix proteins, such as amelogenins on at least parts of the bone graft surface is achieved after 5-120 minutes, such as after 5-60 minutes, 10-30 minutes, 5-30 minutes, or 15-60 minutes. In one embodiment, sufficient aggregation and/or precipitation and agglomeration of enamel matrix proteins, such as amelogenins on at least parts of the bone graft surface is achieved no later than 120 minutes after bringing the bone graft and the composition according to the present invention into contact, such as no later than 5, 10, 15, 20, 25, 30, 45, 60, 90 or 120 minutes thereafter.
  • bioactivating encompasses enhancing bioactivity and improving osteoconduction of a bone graft material.
  • a bioactivated bone graft material is a bone graft material with enhanced bioactivity and/or improved osteoconduction promoting properties. As a result of said bioactivation, said bone graft material will display enhanced biocompatibility after implantation.
  • the bone graft material being bioactivated in a process as disclosed in the present document is selected from the group consisting of natural bone, synthetic bone and scaffolds.
  • the bone graft material being bioactivated in a process as disclosed in the present is a bovine based xenograft, such as BioOss.
  • the bone graft material is natural bone.
  • the bone graft material is synthetic.
  • the present invention is based on the surprising finding that stable and continuous aggregation of EMD and in particular formation of amelogenin aggregates, precipitates and/or agglomerates is dependent not only on the pH and viscosity of the EMD containing composition, but also depends on the porosity of the bone graft material in itself.
  • both Bio-Oss® and Oss permit a stable and continuous aggregation of EMD, even if this effect is more pronounced for Bio-Oss. This may be due to the lower porosity of Oss, leading to the EMD particles, which do adsorb and are integrated by the scaffold granules in the experimental set-up, do so at a lesser speed.
  • porosity is defined by density and pore size of a given bone graft material.
  • the size and the interconnectivity of the pores in bone graft materials are critical factors for a successful clinical outcome. These properties influence diffusion of nutrients and oxygen, attachment, migration, and differentiation of cells, and tissue ingrowth, which are crucial steps in bone augmentation.
  • two types of pores have to be distinguished: 1) macropores (diameter > 100 ⁇ m) which confer osteoconductive properties on bone substitutes and favor cell colonization; it has even been proposed that the influence of the macropore size is greater than that of macroporosity percentage, 2) micropores (diameter ⁇ 100 ⁇ m) which are important for the penetration and adhesion of macromolecules and tissue fluids.
  • the total volume of all kinds of pores present in a material defines the total porosity.
  • a xenogenic bone substitute material made from bovine bone and consisting of small and compact natural apatite crystals has a particularly high capacity to be bioactivated with a low viscosity composition comprising EMD.
  • a preferred bone graft material has a structure similar to the bone it is prepared from.
  • the bone graft material to be bioactivated or the bioactivated bone graft material is a bone graft material that has a total porosity of about 70-98%, such as about 70%, 75%, 80%, 85%, 90.0 %, 91.2 %, 92%, 93% or 95%, such as at least 70%, 75%, 80%, 85%, 90.0 %, 91.2 %, 92%, 93% or 95%
  • the average pore size of a preferred bone graft material to be bioactivated, or of the bioactivated bone graft material according to the present invention, expressed as average pore diameter, is selected from the group consisting of pore sizes between 0.1 ⁇ m and 1000 ⁇ m, such as about 100-800 ⁇ m, 200-800 ⁇ m, 250 - 1000 ⁇ m , 1-10 ⁇ m and 100-500 ⁇ m.
  • a bone graft materials suitable for use in accordance with the present document include, but is not limited to, the porous biphasic synthetic bone-graft substitute in granulated form, denoted Oss, consisting of biphasic calcium phosphate, a composite of 10% hydroxyapatite and 90% ⁇ -tricalcium phosphate.
  • Oss the porous biphasic synthetic bone-graft substitute in granulated form
  • the pore size is 0.1 - 1000 ⁇ m.
  • the total porosity in this material is about 50-85%, such as 65 ⁇ 15% or 70 ⁇ 15%.
  • BoneCeramic® (Straumann AG, Basel, Switzerland), which a synthetic bone-graft substitute designed for augmenting bone consisting of biphasic calcium phosphate with a composite of 60% hydroxyapatite and 40% ⁇ -tricalcium phosphate.
  • BoneCeramic® is 90% porous with interconnected pores of 100-500 ⁇ m in diameter.
  • a suitable bone graft material is Botiss maxresorb® (Botiss dental GmbH, Berlin, Germany) which is a synthetic bone-graft substitute designed for augmenting bone. It consists of biphasic calcium phosphate with a composite of 60% hydroxyapatite and 40% ⁇ -tricaicium phosphate. Maxresorb® is 80% porous with interconnected pores of 200-800 ⁇ m in diameter; micropores have a diameter of 1-10 ⁇ m.
  • a further example of a suitable bone graft material are xenogenic hydroxyapatite particles (BioOss®, Geistlich Biomaterials, Wolhusen, Switzerland), a natural product of bovine origin, which is deproteinized and sintered. The total porosity of this material is 70 - 75% with a particle size of 250 - 1000 ⁇ m.
  • a process for preparing a bioactivated bone graft material for improved bone regeneration comprising obtaining a porous bone graft material, coating and/or soaking and/or filling said bone graft material with a composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT), and optionally lyophilizing said bioactivated bone graft material.
  • a composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT), and optionally lyophilizing said bioactiv
  • a kit comprising a) a low viscosity composition comprising enamel matrix derivative (EMD) together with b) a porous bone graft material, to be combined immediately before the placing of the bioactivated bone graft material by the skilled practitioner.
  • a bioactivated bone graft material soaked in and/or coated with a low viscosity composition comprising enamel matrix derivative (EMD), is provided ready for use.
  • a kit for preparing a bioactivated bone graft material for improved bone regeneration, comprising at least two components;
  • a bioactivated bone graft material according to according to the present invention can be used in medicine.
  • the present invention discloses a bioactivated bone graft material according to the present invention for use in improving bone regeneration.
  • the present invention further relates to the use of a composition comprising isolated enamel matrix proteins at a concentration of 0.3 mg/ml-100 mg/ml in a suitable pharmaceutical carrier, said composition having a pH between pH 3.8 - 4.2, and a viscosity of less than 50 m Pa ⁇ s at 22°C (RT), such as between 30-1.0 mPa ⁇ s at 22°C (RT) for manufacturing a bioactivated bone graft material according to the present invention for improving bone regeneration.
  • RT viscosity
  • Also envisioned is a method for improving bone regeneration in a patient in need thereof comprising implanting a bioactivated bone graft material according to the present invention into a patient in need thereof.
  • Bio-Oss® is purchased from Geistlich Biomaterials, and it consists of granules having diameters of 0.25 to 1mm.
  • Oss is mainly constituted by hydroxyapatite grains of the same size range.
  • the respected ratio of Bio-Oss® or Oss was 1mg for 1 ⁇ L of EMD.
  • the selected ProteoStat R Protein Aggregation Assay by Enzo has been mixed with the specific dye and Ultrapur water in the following proportions: 10 ⁇ L Dye + 20 ⁇ L Assay + 170 ⁇ L H20. After putting 2 ⁇ L of this solution and 100 ⁇ L of EMD at a concentration of 31mg/mL in each recipient, also 100mg of Bio-Oss® were added in half of the cases.
  • parameters related to the characteristics of the dye have to be entered in the program, as the excitation and emission wavelengths and bandwidths, the gain, the integration time and the number of cycles that have to be done.
  • the Bio-Oss® or Oss granules have to be separated from the liquid solution and left to dry. Later, in order to reduce charging effects and improve the quality of the image, the samples have to be gold-coated by a process called metallization. In other words, a thin layer of gold is deposited onto the sample and will permit to evacuate the electrons of it.
  • Figure 2 shows the pH variation of solutions of EMD mixed with Bio-Oss® at initial pH 4.37, 3.87, 3.68, and 3.40 with and without arginine. It is noticeable that the samples at initial pH 4.37 reach a value of 6 in less than 10 minutes, which causes EMD precipitation due to the vicinity to the isoelectric point of amelogenin (pH 6.8). The solutions at initial pH 3.87 reach the precipitation after 200 minutes, while the two lower pH solutions precipitate only after 6 days (not presented in the graph). Whether or not arginine is added to the solutions does not seem to really influence the velocity at which the pH evolves. As the precipitation of the EMD particles has to occur within reasonable times for the application, only the solutions at initial pH 4.37 and 3.87 will be considered in the following results regarding Bio-Oss®.
  • Figure 3 shows the fluorescence intensity of EMD in acetic acid with and without Bio-Oss® at different initial pH.
  • the solutions containing Bio-Oss® exhibit a higher intensity than the samples without it.
  • In presence of Bio-Oss® aggregation is fast since the pH increase is favored up to precipitation, and this explains the higher intensity of the fluorescence signal.
  • samples without Bio-Oss® seem not to aggregate, because there signal intensity remains constant on the overall time scale.
  • Figure 4 shows the fluorescence intensity of EMD in acetic acid and arginine with and without Bio-Oss® at different initial pH. It is noticeable that the solution at initial pH 4.27 containing Bio-Oss® has a higher intensity than the ones without, while the solution at initial pH 3.87 has the lower signal intensity. The graph also shows that by increasing the pH of the solutions, the signal intensity raises, which means that more aggregation occurs. Compared to Figure 3 , the addition of arginine does that the signal intensity for the solutions at initial pH 4.27 and 3.87 with Bio-Oss® are decreasing.
  • Figure 9 is presented an image of EMD solution at initial pH 4.27 with Bio-Oss® granules obtained by scanning electron microscopy. It is possible to observe EMD aggregates with a globular shape that covers most of the surface of the granules after precipitation.
  • Figure 10 shows the pH variation of solutions of EMD mixed with Oss at pH 4.41, 3.93, 3.76, and 3.31 with and without arginine. It is noticeable that between two and three hours, the samples at pH 4.41 are the first to reach a pH value of 6, which causes EMD precipitation due to the vicinity to the isoelectric point of amelogenin (pH 6.8). Even if it is not shown on the graph, the solutions at pH 3.93, 3.76, and 3.31, reached the pH value of 6 respectively after 15 hours, 5 days, and 8 days. When arginine is added to the solutions, it increases the velocity at which the pH evolves. As in the case of Bio-Oss®, the precipitation of the EMD particles has to occur within reasonable times, this is why only the solution at pH 4.41 will be considered in the following results regarding Oss granules.
  • Figure 11 shows the fluorescence intensity of EMD in acetic acid with and without Oss granules at different pH.
  • the signal intensity raises, meaning that more aggregates are present.
  • the solutions containing the scaffold exhibit a higher intensity than the samples without it, because aggregation is fast since the pH increase is again favored up to precipitation.
  • the EMD in acetic acid solutions have a constant signal in time, hence no aggregation occurs, while the ones containing Oss are undergoing a lot of oscillations.
  • Figure 12 shows the fluorescence intensity of EMD in acetic acid and arginine with and without Oss at different pH. It is noticeable that the solutions containing Oss have a higher intensity signal than the ones without. As in Figure 11 , the solutions without Oss have a signal intensity that stays almost constant in time, whereas the solutions with Oss show an oscillating signal. As a general trend for the solutions without Oss, when the pH of the solution increased, the fluorescence signal is higher. Finally, when comparing with Figure 11 , it is noticeable that the addition of arginine does that the signal intensity values for EMD solutions with Oss are increasing a lot.
  • Figure 14 shows a scanning electron microscopy image of an EMD solution at initial pH 4.41 containing Oss granules. After precipitation, it is possible to observe that there are no EMD particles aggregates that are present on the surface of the granules.
  • EMD Enamel Matrix Derivative
  • the goal of this experiment was to measure the viscosity of a composition comprising active enamel proteins (Osteogain®) in acetic acid.
  • the time for Osteogain® is 1.8 times longer than the time for water. Since the difference in density for Osteogain® and water is neglectable, the viscosity for Osteogain is 1.8 times higher than for water.
  • the viscosity for water at 22°C is 0.9548 cP (according to Handbook of Chemistry and Physics 64th edition table F-38 ) giving the viscosity for Osteogain as 1.7 cP (i.e. 1.7m Pa ⁇ s at 22°C (RT)).
  • the viscosity of Osteogain® is 1.8 times the viscosity of water.
  • the water viscosity at 22°C is 0.9548 cP giving the viscosity of Osteogain as 1.7 cP.
  • the Osteogain® solution is several magnitudes less viscous.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Dispersion Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Materials For Medical Uses (AREA)

Claims (23)

  1. Zusammensetzung zum Bioaktivieren eines Knochentransplantatmaterials, wobei die Zusammensetzung isolierte Schmelzmatrixproteine mit einer Konzentration von 0,3 mg/ml - 100 mg/ml in einem geeigneten pharmazeutischen Träger umfasst, wobei die Zusammensetzung einen pH zwischen pH 3,8-4,2 und eine Viskosität von weniger als 50 mPa·s bei 22 °C aufweist.
  2. Zusammensetzung nach Anspruch 1, wobei die Zusammensetzung einen pH zwischen pH 3,8-4,2 und eine Viskosität zwischen 30-1 mPa·s bei 22 °C aufweist.
  3. Zusammensetzung nach Anspruch 1 oder 2, wobei die Zusammensetzung einen pH zwischen pH 3,8-4,2 und eine Viskosität zwischen 2,5-1,2 mPa·s bei 22 °C aufweist.
  4. Zusammensetzung nach einem der vorstehenden Ansprüche, wobei die Zusammensetzung einen pH von 4,0 und eine Viskosität von 1,7 mPa·s bei 22 °C aufweist.
  5. Zusammensetzung nach einem der vorstehenden Ansprüche, wobei der pharmazeutische Träger nicht Propylenglykolalginat ist.
  6. Zusammensetzung nach einem der vorstehenden Ansprüche, wobei der pharmazeutische Träger Essigsäure ist.
  7. Zusammensetzung nach einem der vorstehenden Ansprüche, wobei die Konzentration der Schmelzmatrixproteine 29-31 mg/ml, beispielsweise 29, 30 oder 31 mg/ml beträgt.
  8. Verwendung einer Zusammensetzung nach einem der Ansprüche 1-7 in einem In-vitro-Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials, das für verbesserte Knochenregeneration vorgesehen ist.
  9. Verwendung einer Zusammensetzung nach Anspruch 8, wobei die Gesamtporosität des Knochentransplantatmaterials mindestens 70 % beträgt.
  10. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration, umfassend
    a) Erhalten eines porösen Knochentransplantatmaterials,
    b) Beschichten und/oder Durchtränken und/oder Füllen des Knochentransplantatmaterials mit einer Zusammensetzung, umfassend isolierte Schmelzmatrixproteine mit einer Konzentration von 0,3 mg/ml - 100 mg/ml in einem geeigneten pharmazeutischen Träger mit einem pH zwischen pH 3,8-4,2 und einer Viskosität von weniger als 50 mPa·s bei 22 °C, und
    c) gegebenenfalls Lyophilisieren des Knochentransplantatmaterials.
  11. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach Anspruch 10, wobei die Gesamtporosität des Knochentransplantatmaterials mindestens 70 % beträgt.
  12. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach einem der Ansprüche 10-11, wobei die Gesamtporosität des Knochentransplantatmaterials zwischen 70-98 %, beispielsweise 70 %, 75 %, 80 %, 85 %, 90,0 %, 91,2 %, 92 %, 93 % oder 95 % beträgt.
  13. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach einem der Ansprüche 10-12, wobei der pH der Zusammensetzung vor dem Mischen der Komponenten a) und b) zwischen pH 3,8-4,2 liegt.
  14. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach einem der Ansprüche 10-13, wobei die Zusammensetzung, umfassend isolierte Schmelzmatrixproteine, die in Schritt b) verwendet werden, isolierte Schmelzmatrixproteine mit einer Konzentration von 29-31 mg/ml, beispielsweise mit einer Konzentration von 29, 30 oder 31 mg/ml umfasst.
  15. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach einem der Ansprüche 10-14, wobei der geeignete pharmazeutische Träger der Zusammensetzung, umfassend isolierte Schmelzmatrixproteine, die in Schritt b) verwendet werden, Essigsäure ist.
  16. Verfahren zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach einem der Ansprüche 10-15, wobei das Knochentransplantatmaterial ausgewählt ist aus der Gruppe, bestehend aus natürlichem Knochen, synthetischem Knochen und Stützgewebe.
  17. Bioaktiviertes Knochentransplantatmaterial zur verbesserten Knochenregeneration, das unter Verwendung eines Verfahrens nach einem der Ansprüche 10-16 hergestellt wird und worin mindestens 50-90 % der isolierten Schmelzmatrixproteine, die in und/oder auf dem bioaktivierten Knochentransplantatmaterial umfasst sind, zusammengesetzte Amelogenin-Nanokugeln sind, die in einem um 1500 nm zentrierten Hauptpeak, gemessen mit DLS, ausfallen.
  18. Bioaktiviertes Knochentransplantatmaterial zur verbesserten Knochenregeneration nach Anspruch 17, wobei das Knochentransplantatmaterial porös ist.
  19. Bioaktiviertes poröses Knochentransplantatmaterial zur verbesserten Knochenregeneration nach Anspruch 18, wobei das Knochentransplantatmaterial ausgewählt ist aus der Gruppe, umfassend ein Rinderxenotransplantat und ein synthetisches Knochentransplantatmaterial.
  20. Bioaktiviertes poröses Knochentransplantatmaterial zur verbesserten Knochenregeneration nach einem der Ansprüche 18-19, wobei das Knochentransplantatmaterial in Form von Granulaten vorliegt.
  21. Kit zur Herstellung eines bioaktivierten Knochentransplantatmaterials zur verbesserten Knochenregeneration nach einem der Ansprüche 18-20, das mindestens zwei Komponenten umfasst;
    a. ein poröses Knochentransplantatmaterial, und
    b. eine Zusammensetzung, umfassend isolierte Schmelzmatrixproteine mit einer Konzentration von 0,3 mg/ml - 100 mg/ml in einem geeigneten pharmazeutischen Träger, wobei die Zusammensetzung einen pH zwischen pH 3,8-4,2 und eine Viskosität von weniger als 50 mPa·s bei 22 °C aufweist.
  22. Bioaktiviertes Knochentransplantatmaterial nach einem der Ansprüche 17-20 zur Verwendung in der Medizin.
  23. Bioaktiviertes Knochentransplantatmaterial zur verbesserten Knochenregeneration nach einem der Ansprüche 17-20 zur Verwendung für die Verbesserung der Knochenregeneration.
EP15738878.6A 2014-07-09 2015-07-09 Emd mit niedriger viskosität Active EP3166629B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE1450883 2014-07-09
PCT/EP2015/065694 WO2016005495A1 (en) 2014-07-09 2015-07-09 Low viscosity emd

Publications (2)

Publication Number Publication Date
EP3166629A1 EP3166629A1 (de) 2017-05-17
EP3166629B1 true EP3166629B1 (de) 2019-04-24

Family

ID=53673922

Family Applications (1)

Application Number Title Priority Date Filing Date
EP15738878.6A Active EP3166629B1 (de) 2014-07-09 2015-07-09 Emd mit niedriger viskosität

Country Status (3)

Country Link
US (1) US10525167B2 (de)
EP (1) EP3166629B1 (de)
WO (1) WO2016005495A1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112770718A (zh) * 2018-08-30 2021-05-07 华盛顿大学 牙科护理的组合物和方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60140191D1 (de) 2000-06-20 2009-11-26 Straumann Inst Ag Zusammensetzungen mit matrixproteinen zur regeneration von dentin
AU7437901A (en) 2000-06-21 2002-01-02 Biora Bioex Ab Matrix protein compositions for inhibition of epithelial cell growth
AU2002334515B2 (en) 2001-09-14 2007-01-04 Institut Straumann Ag Enamel matrix protein compositions for modulating immune response
US20100233783A1 (en) * 2005-03-24 2010-09-16 Straumann Holding Ag Novel method for protein purification
CN103153334B (zh) 2010-10-15 2016-11-16 斯特劳曼控股公司 包含pga的新的emd制剂
EP3094308B1 (de) 2014-01-17 2018-12-26 Straumann Holding AG Prozess zur herstellung zahnemaillematrixproteine von erhöhter stabilität

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
US20180169303A1 (en) 2018-06-21
WO2016005495A1 (en) 2016-01-14
EP3166629A1 (de) 2017-05-17
US10525167B2 (en) 2020-01-07

Similar Documents

Publication Publication Date Title
Subramaniam et al. Hydroxyapatite-calcium sulfate-hyaluronic acid composite encapsulated with collagenase as bone substitute for alveolar bone regeneration
Kolk et al. Current trends and future perspectives of bone substitute materials–from space holders to innovative biomaterials
Lee et al. Injectable biodegradable gelatin-methacrylate/β‐tricalcium phosphate composite for the repair of bone defects
Carvalho et al. Bioactive glass nanoparticles for periodontal regeneration and applications in dentistry
US8690874B2 (en) Composition and process for bone growth and repair
US20080063681A1 (en) Therapeutic bone replacement material
Struillou et al. Treatment of periodontal defects in dogs using an injectable composite hydrogel/biphasic calcium phosphate
ES2501965T3 (es) Preparación para la regeneración de defectos óseos posoperatorios y postraumáticos
Weng et al. The effects of recombinant human growth/differentiation factor-5 (rhGDF-5) on bone regeneration around titanium dental implants in barrier membrane-protected defects: a pilot study in the mandible of beagle dogs.
Sarin et al. Bioactive glass: a potential next generation biomaterial
Sandor et al. Bone regeneration of the cranio-maxillofacial and dento-alveolar skeletons in the framework of tissue engineering
Kumar et al. Journey of bone graft materials in periodontal therapy: A chronological review
EP1009333A1 (de) Formbare, bioaktive zusammensetzung
EP3166629B1 (de) Emd mit niedriger viskosität
NL2007850C2 (en) Injectable calcium phosphate cement comprising glucono-delta-lactone.
Dziak et al. Nanoceramics for bone regeneration in the oral and craniomaxillofacial complex
Macedo et al. Vital bone formation after grafting of autogenous bone and biphasic calcium phosphate bioceramic in extraction sockets of rats: Histological, histometric, and immunohistochemical evaluation
Sheikh et al. Bone grafting
Naredla et al. Potential applications of coral sand in bone healing and drug delivery
Kürkcü et al. Histomorphometric evaluation of implants coated with enamel or dentine derived fluoride-substituted apatite
KR20210145130A (ko) 프롤린-풍부 펩타이드를 포함하는 개선된 뼈 임플란트 매트릭스 및 그것의 제조 방법
Saeed et al. Fine calcified human dentin particles grafts in experimental bone defects in rabbit femur accelerate bone healing and maturation
Park et al. Bone regeneration capacity of two different macroporous biphasic calcium materials in rabbit calvarial defect
Komath et al. Designing Bioactive Scaffolds for Dental Tissue Engineering
NL2030793B1 (en) Malleable bone repair material

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20170208

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20171123

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20181206

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1123336

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190515

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602015028891

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20190424

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190724

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190824

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190724

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190725

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1123336

Country of ref document: AT

Kind code of ref document: T

Effective date: 20190424

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190824

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602015028891

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20190724

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

26N No opposition filed

Effective date: 20200127

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20190731

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190724

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190731

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190709

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190731

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20190709

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20150709

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190424

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20230801

Year of fee payment: 9

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20240719

Year of fee payment: 10